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Cyclin D1 overexpression is associated with poor prognosis in oropharyngeal cancer Lin, Rui J; Lubpairee, Tarinee; Liu, Kelly Y; Anderson, Donald W; Durham, Scott; Poh, Catherine F Mar 19, 2013

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ORIGINAL RESEARCH ARTICLE Open AccessCyclin D1 overexpression is associated with poorprognosis in oropharyngeal cancerRui Jun Lin1, Tarinee Lubpairee2, Kelly Y Liu1, Donald W Anderson1, Scott Durham1 and Catherine F Poh2*AbstractObjectives: To determine the biological characteristics of oropharyngeal squamous cell carcinoma (OpSCC) andrelated outcome.Design: Retrospective study.Methods: Patients (N=60) with primary OpSCC from 2000 to 2005 were retrospectively identified from Pathologydatabase and the outcome was confirmed through chart review. Among these, 41 biopsy samples with enoughtissues were retrieved to construct a tissue microarray for detection of the presence of high-risk humanpapillomavirus (HPV) using Chromogenic in situ hybridization (CISH) as well as the expression of p16 and cyclin D1using immunohistochemistry.Main outcome measures: Disease-free survival.Results: Among 60 patients, 39 (65%) patients had no recurrence or died without disease at the last follow-up(disease-free survival or Group 1), and 21 (35%) patients had persistent disease or died of disease (progression-freesurvival or Group 2). Although follow-up time was twice as long in group 1 (4.7 ± 2.2 vs. 2.0 ± 1.6 years; P < 0.0001),there was no difference between the 2 groups in age, gender, smoking/alcohol habits, TNM staging and treatmentmodalities. Among those 41 cases with available tumour tissues, there was no difference in HPV status and p16expression between the 2 groups but a significant difference in cyclin D1 expression (P = 0.05). Using Kaplan-Meirsurvival analysis and log-rank test, cyclin D1 overexpression was highly associated with a poor prognosis whencomparing time to outcome (P < 0.0001).Conclusion: Cyclin D1 overexpression is a potential prognostic marker of OpSCC.Keywords: Oropharyngeal squamous cell carcinoma (OpSCC), Human papillomavirus (HPV), p16, Cyclin D1, Diseasefree survival, PrognosisThe incidence of oropharyngeal squamous cell carci-noma (OpSCC), particularly those arising in the base ofthe tongue and in the tonsillar region, rises every yearbetween 1973 and 2004 [1]. This increasing trend occursdespite the decrease in the incidence of oral cavity, la-ryngeal and hypopharyngeal cancers as well as the de-creased prevalence in smoking, which is a primary riskfactor for these cancers [2]. Molecular studies have iden-tified human papillomavirus (HPV) as a causative agentin 60% to 80% of patients with OpSCC [3]. HPV-16 ac-counts for a large majority of HPV-positive OpSCC (80-90%) compared to other oncogenic types such as HPV-18, 31 or 33 [4,5].HPV-positive OpSCCs have distinct risk factors andmolecular profiles. These tumours are thought to be as-sociated with certain types of sexual behavior, but not totobacco smoking or alcohol use. On a molecular level,almost all HPV-positive tumours express wild-type p53tumour suppressor genes. Expression of HPV viral E6and E7 oncoproteins inactivates p53 and the retinoblast-oma protein pRb. p16 protein expression is subsequentlyelevated due to loss of the pRb negative feedback loop[6]. p16 then inhibits cyclinD1-CDK4/6 (cyclin-dependent kinase 4/6) complexes, which are importantregulators for the cell cycle to progress from the G1phase to the S phase. As a result, HPV-positive cancers* Correspondence: CPoh@bccancer.bc.ca2Integrative Oncology, BC Cancer Agency & Research Centre, Vancouver, BC,CanadaFull list of author information is available at the end of the article© 2013 Lin et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Lin et al. Journal of Otolaryngology - Head and Neck Surgery 2013, 42:23http://www.journalotohns.com/content/42/1/23are thought to be associated with downregulation of cyc-lin D1 expression [7,8]. In other head and neck cancersthat are not typically related to HPV, cyclin D1 over-expression has been linked with poor outcome, particu-larly in hypopharyngeal cancers [9]. However there isnot a large amount of evidence suggesting its prognosticsignificance in oropharyngeal cancer.Given the worldwide growing epidemic of HPV-associated oropharyngeal cancers, characterization ofHPV-associated molecular markers is becoming increas-ingly important for determining their impact on theprognosis of disease and for guiding the development ofnew therapeutic interventions. The objective of ourstudy was to determine the association of cyclin D1 andHPV status in OpSCC and its relevance on patient out-come. Since cyclin D1 overexpression has been asso-ciated with a poor prognosis in cancers of other headand neck regions, we hypothesize it is associated with apoor prognosis in oropharyngeal cancer.MethodsPatient and tumour tissueThis is a retrospective case series. The study hasobtained approval from the Research Ethics Board at theUniversity of British Columbia and has been in com-pliance with research conduct guidelines at this institu-tion. A total of 60 patients (n = 60) with primaryOpSCC from the year 2000 to 2005 were retrospectivelyidentified from the Vancouver General Hospital Pa-thology database. This duration was chosen so that allpatients have had at least 5 years of follow-up after theirinitial treatment. These patients were confirmed to haveno previous history of cancer and their outcome infor-mation were collected through electronic chart reviewfrom the patient information database at the BritishColumbia Cancer Agency (BCCA). Demographic andclinical data were also collected from chart review, in-cluding age, gender, smoking status, alcohol intake,treatment received, follow-up time and survival status.Tumour staging using the TNM classification [10] was de-termined by review of clinical notes, radiology reports,surgical notes and pathology reports. Among the includedpatients, 41 (68%) formalin-fixed biopsy specimens withenough tissues were retrieved from the pathology archivefor the construction of tissue microarrays.Tissue microarray (TMA) constructionTissue microarray was constructed as previously described[11]. The most representative areas of tumours werechosen by the pathologist (CFP). Tissue cores of 0.6 mmin diameter from these representative areas of formalin-fixed paraffin-embedded (FFPE) tumour blocks were takenby a manual tissue arrayer (Beecher Instrument, SI, USA).The cores were then transferred in duplicates to therecipient TMA block. The recipient blocks were then se-rially cut into 4 μm tissue sections for chromogenic in situhybridization and immunohistochemistry.Chromogenic in situ Hybridization (CISH) for HPVhigh-risk subtypes and HPV-16/18High-risk HPV subtypes and specifically HPV subtypes 16as well as 18 in FFPE TMA sections were detected byusing chromogenic in situ hybridization (CISH). TwoHPV probes were examined in this study: HPV-HR (cock-tail for high-risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56,58, 59, and 68, DAKO, Denmark), and HPV-16/18(DAKO, Denmark). In brief, each of the tissue sectionswere deparaffinized, rehydrated and pretreated with targetretrieval solution (DAKO) for 20–40 minutes at 97°C.After antigen retrieval with Proteinase K (Sigma-Aldrich,Canada) and blocking, the slides were hybridized with abiotinylated HPV-HR probe and HPV-16/18-specificprobe. After initial binding of streptavidin horseradish per-oxidase complex to the probe, signal amplification wasperformed using biotinyl tyramide. Positive hybridizationsignals were visualized by adding the chromogenic sub-strate diaminobenzidine (DAB). SiHa and SCC-9 cell lineswere used as a positive and negative control, respectively.Slides were scored positive for HPV if a nuclear, punctuatepattern of signals was observed in > 60% of the tumournuclei, which was indicative of integrated HPV DNA intothe host genome. The scoring was performed by the samepathologist (CFP).Immunohistochemistry (IHC) for p16 and cyclin D1IHC was performed on TMA tissue sections that weredeparaffinized. After target retrieval, sections were incu-bated with primary antibodies for p16 (Roche mtm la-boratories AG, Heidelberg, Germany) [12] and cyclin D1(Cell Signaling, Ontario, Canada) [13] with duration andtemperature specified for each marker (Table 1). Thesections were then incubated with the EnVision+ System(DAKO Corporation) for cyclin D1 for detection ofantigen-antibody reactions. Sections were visualized withDAB and counterstained with Mayer’s hematoxylin. Thestaining intensity was scored as a percentage of stainedtumour cells (0% - 100%). Tumour cells with a greaterthan or equal to 50% staining percentage were scored as“positive”. Otherwise they were scored as “negative”. Thescoring was again performed by the same pathologist(CFP).Statistical analysisAll statistical analyses were performed using SPSS/PASW Statistics (version 19, SPSS Inc., Chicago, IL).The Fisher’s exact test was used to compare dichoto-mous (e.g., gender, alcohol consumption and N staging)or other categorical (e.g., smoking status, T staging,Lin et al. Journal of Otolaryngology - Head and Neck Surgery 2013, 42:23 Page 2 of 7http://www.journalotohns.com/content/42/1/23TNM staging, and treatment) variables. Student’s t-testwas used to compare parametric variable (e.g., age andfollow-up time). Disease-free survival was estimatedusing the Kaplan-Meier method with log-rank test fordetermining statistical significance. Disease-free survivalwas defined as time from the day of cancer diagnosis tothe date of death from OpSCC or to the date of lastfollow-up. Multivariate analysis was not performed giventhe small sample size of the study. A P value of 0.05 orless was considered statistically significant.ResultsPatient populationSeventy-seven patients were initially identified to have pri-mary oropharyngeal squamous cell carcinoma from theyear 2000 to 2005 from the Pathology database of theVancouver General Hospital. Seventeen patients were ex-cluded from the study after reviewing the electronic chartsat the BC Cancer Agency. Reasons of exclusion included(1) primary tumours not at oropharyngeal site (n = 10);(2) no definitive treatment received due to patient refusalor palliation (n = 6); and (3) loss of follow-up (n = 1). Atotal number of 60 patients were included for the finalanalysis. The patient characteristics are shown in Table 2.The mean follow-up time was 4 ± 2.4 years. The patientswere categorized into two outcome groups based on dis-ease (OpSCC) status after treatment: Group 1 representeddisease-free survival; Group 2 represented progression-free survival or disease-specific deaths. Among the 60 pa-tients, 39 (65%) had no recurrence or died without disease(Group 1) and 21 (35%) had persistent disease or died ofdisease (Group 2). There was no significant differenceamong patient characteristics including age, smoking, al-cohol intake, and TNM staging between the two outcomegroups (Table 2). Follow-up time was significantly longerfor Group 1 compared to Group 2 (4.7 ± 2.2 years vs.2.0 ± 1.6 years; P < 0.0001).HPV status and p16 expressionOf the 60 patients included in the study, 41 had sufficienttumour tissues for analysis. Among these, 56% (n= 23)tested positive for high risk HPV DNA (HPV-HR) and66% (n = 27) showed p16 overexpression. All cases thatwere HPV-HR-positive were also p16-positive (see exam-ples in Figure 1). None of the tumours were HPV-positiveand p16-negative. Approximately 9.8% of tumours werefound to be HPV-negative and p16-positive. The sensitiv-ity, specificity, positive predictive value and negative pre-dictive value using p16 for the presence of HPV-HR DNAdefined by CISH approach were 0.85, 1.0, 1.0, and 0.78, re-spectively. Among those cases that were positive for HPV-HR (n = 23), 68% were positive for HPV-16/18 DNA.While 7 out of 7 tumours from non-smokers were positivefor p16, interestingly 20 out of 32 tumours from smokers(62.5%) were also positive for p16 (P = 0.08).There was no difference in HPV status and p16 ex-pression between Groups 1 and 2 (Table 3). However,disease-free survival was improved in tumours that werepositive for HPV-HR (P =0.05, Figure 2A) compared totumours that were positive for HPV-16/18 (P =0.38,Figures 2B). Although it was not statistically significant,the p16-positive group showed better disease-free sur-vival than the p16-negative group (P = 0.08; Figure 2C).Cyclin D1 expression and disease-free survivalTwelve tumours were cyclin D1-positive (12/41, 29%)and twenty-nine tumours were cyclin D1-negative (29/41, 71%). Among the cyclin D1-negative tumours, 21(78%) were positive for p16, a currently accepted surro-gate marker for the presence of HPV DNA. For thosetumours that were cyclin D1-positive (n = 14), only 43%(n = 6) were positive for p16. Using Fisher’s exact test,there was no difference in cyclin D1 expression betweenthe two outcome groups (Table 3). However, the im-proved disease-free survival over time was significant inthe cyclin D1-negative group using the log-rank analysis(P < 0.0001, hazard ratio 18.0, 95% confidence interval4.37 to 74.03; Figure 2D).DiscussionCyclin D1 overexpression has been reported in squa-mous cell carcinoma in a variety of head and neck sub-sites including the larynx, hypopharynx and tongue[9,14,15]. Its prognostic significance in OpSCC has beencontradictory [16-22]. Our study has demonstrated cyc-lin D1 overexpression is associated with a poor prog-nosis in primary OpSCC. Cyclin D1 is a well-knownoncogene in a variety of human cancers. It is a key regu-lator of cell proliferation. It promotes cell cycle progres-sion through G1 phase by forming complexes withCDK4 and CDK6, which in turn phosphorylate the re-tinoblastoma protein pRb [23]. This causes pRb toTable 1 Antibodies used in immunohistochemistry for p16 and cyclin D1Marker Dilution Incubation Staining pattern PositivecontrolNegativecontrolCatalogue numberp16 Ready touseRoom temperature,30 minNuclear andcytoplasmicSiHa Muscle CINtecW Histology Kit (Roche mtm laboratories AG,Heidelberg, Germany)Cyclin D1 1:35 4°C, Overnight Nuclear SCC-9 Muscle Cyclin D1 (92G2) Rabbit mAb #2978 (Cell Signaling,Ontario, CA)Lin et al. Journal of Otolaryngology - Head and Neck Surgery 2013, 42:23 Page 3 of 7http://www.journalotohns.com/content/42/1/23release the E2F transcription factor, which then activatesgenes essential for cell cycle progression from G1 phaseto S phase, during which cells proliferate [24]. It hasbeen suggested that cyclin D1 plays a role in the latephase of tumour progression given its correlation withlymph node metastases, histological aggressiveness andpoor prognosis [9].Cyclin D1 overexpression was higher in patients withadverse outcomes in our study, but the difference wasnot statistically significant. With the log-rank analysis,Table 2 Comparison of patient characteristics between two outcome groupsTotal (N = 60) Group 1a (N=39, 65%) Group 2a (N = 21, 35%) P valueMean age at diagnosis (yr ± SD) 60.4 ± 11.7 59.8 ± 11.3 61.4 ± 12.6 0.63GenderMale 46 (77) 30 (77) 16 (76)Female 14 (23) 9 (23) 5 (24) 1Smoking HabitNon-smokers 14 (23) 10 (26) 4 (19) 0.78Former smokers (quit > 5 yr) 15 (25) 9 (23) 6 (29)Current smokers 29 (48) 20 (51) 9 (43)Unknowne 2 (3) 0 (0) 2 (10)AlcoholbNever or light 51 (85) 34 (87) 17 (81) 1Heavy 7 (12) 5 (13) 2 (10)Unknowne 2 (3) 0 (0) 2 (10)T staging1 25 (42) 19 (49) 6 (29) 0.442 15 (25) 9 (23) 6 (29)3 12 (20) 6 (15) 6 (29)4 3 (5) 2 (5) 1 (5)Unknowne 5 (8) 3 (8) 2 (10)N stagingN0 7 (12) 6 (15) 1 (5) 0.4N+ 51 (85) 32 (82) 19 (90)Unknowne 2 (3) 1 (3) 1 (5)TNM stagingc1 2 (3) 2 (5) 0 (0) 0.612 4 (7) 3 (8) 1 (5)3 11 (18) 6 (15) 5 (24)4 41 (68) 27 (69) 14 (67)Unknowne 2 (3) 1 (3) 1 (5)Treatment informationSurgery (S/T) only 3 (5) 2 (5) 1 (5) 0.12Radiation (XRT) onlyd 30 (50) 18 (46) 12 (57)Chemotherapy only 11 (18) 5 (13) 6 (29)Combined S/T and XRT 16 (27) 14 (36) 2 (10)Mean Follow-Up Time (yr ± SD) 4 ± 2.4 4.7 ± 2.2 2 ± 1.6 < 0.0001aOutcome: Group 1, those survived with no disease or died due to other cause (disease-free survival); Group 2, those alive with disease or died of disease(progress-free survival or disease-specific deaths).bHeavy alcohol intake was defined as > 3 drinks/day for males and > 2 drinks/day for females.cTNM staging is based on the American Joint Committee on Cancer (AJCC) Cancer Staging Manual. 7th ed 2010.dRadiation only include radiation only with/without induction chemotherapy.eMissing values (Group 1 (N): Group 2 (N)): Smoking Habit (0:2), Alcohol (0:2), T-staging (3:2); Nodal Status (1:1); TNM staging (1:1).Lin et al. Journal of Otolaryngology - Head and Neck Surgery 2013, 42:23 Page 4 of 7http://www.journalotohns.com/content/42/1/23however, the difference in disease-free survival over timewas statistically significant between the cyclin D1-positive and cyclin D1-negative groups. Although thisstudy had a small sample size, we fitted a multivariateCox Proportional Hazard model to examine the poten-tial joint and interaction effects among markers (datanot shown). The analysis showed that cyclin D1 over-expression was the only and independent variable de-monstrating significance in predicting poor outcome indisease-free survival. Despite the small number of pa-tients in our study, our finding strongly suggest thatcyclin D1 could potentially be a useful prognosticmarker for OpSCC.In our study, HPV DNA was detected in 56% ofOpSCC with the entire 56% being p16-positive. This isconsistent with the reported numbers from previousstudies [25,26]. 9.8% of tumours were p16-positive andHPV-negative, which has also been reported in the li-terature [26]. Overall, our study supports p16 as an ef-fective surrogate marker for high-risk HPV infectionwith high sensitivity and specificity (85% and 100%, re-spectively). Cyclin D1 expression should be down-Figure 1 Examples of a case from Group 1 (favourable outcome group; upper panel, A, B, C) and a case from Group 2 (adverseoutcome group; lower panel, D, E, F). A (diffuse punctate signals) and D (negative) showing CISH staining of HPV-HR DNA; B (diffuse strongcytoplasmic staining and nuclear staining) and E (negative) showing IHC staining of p16 protein; C (negative) and F (diffuse strong nuclearstaining) showing IHC staining of cyclin D1. (Original magnification 100×).Table 3 Comparison of tumour HPV DNA, p16 and cyclin D1 status between outcome groups 1 and 2Total (N=60) Group 1 (N=39, 65%) Group 2 (N=21, 35%) P valueHPV-HRPositive 23 (56) 14 (61) 9 (50) 0.54Negative 18 (44) 9 (39) 9 (50)HPV-16/18Positive 15 (37) 8 (35) 7 (39) 1Negative 26 (63) 15 (65) 11 (61)p16Positive 27 (66) 16 (70) 11 (61) 0.74Negative 14 (34) 7 (30) 7 (39)Cyclin D1Positive 12 (29) 4 (17) 8 (44)Negative 29 (71) 19 (83) 10 (56) 0.09Lin et al. Journal of Otolaryngology - Head and Neck Surgery 2013, 42:23 Page 5 of 7http://www.journalotohns.com/content/42/1/23regulated in HPV-positive head and neck squamouscell carcinoma as a result of pRb suppression [7,24]. Inour study, 78% (N=21) of cyclin D1-negative tumourswere associated with p16 overexpression. This inverserelationship is consistent with the hypothesis that HPVpositivity and subsequent p16 upregulation leads to sup-pression of cyclin D1 expression.Our study has not found any significant associationbetween smoking or alcohol intake and tumour HPVstatus. Interestingly, all the nonsmokers showed p16/HPV positivity but 62.5% of smokers have also shownp16/HPV positivity. This may argue against the currentliterature observation that HPV-positive tumours tendto occur in non-smokers and non-drinkers [27].One of the limitations of this study is that it was aretrospective review, thus it relied on the accuracy ofwritten or dictated medical records. A second limitationis the small sample size. We were only able to identify atotal number of 60 cases of primary oropharyngeal car-cinoma with known outcome from 2000 to 2005. Thuswe may not have enough patients in order to detect astatistical significance in the expression of markers be-tween the two outcome groups. We were also not ableto perform a multivariate analysis due to the small sam-ple size. The next step of this project is to widen oursearch through the BC Cancer Registry in order to iden-tify more primary oropharyngeal cancer cases and toretrieve their tumour tissues for tissue microarray con-struction. A prospective arm of the study is alsounderway to recruit patients with new oropharyngealcarcinoma diagnosis to participate in our investigation.ConclusionOur preliminary results have demonstrated cyclin D1overexpression is a potential prognostic marker of pri-mary oropharyngeal squamous cell carcinoma. Furtherunderstanding of these genetic alterations leading to ma-lignant progression is critical to future secondary pre-vention strategies and therapeutic interventions forpatients with HPV-positive oropharyngeal squamous cellcarcinoma.ConsentWritten informed consent was obtained from patientswho participated in this study for publication of this re-port and any accompanying images.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsRJL, SD and CFP designed the study protocol. RJL was responsible for ethicsapplication, patient chart review, data collection and manuscript write-up. TLand KYL performed tissue microarrays, data collection and statistical analysis.DWA and SD were responsible for recruiting patients. CFP was the principleinvestigator overlooking the entire study. All authors read and approved thefinal manuscript.AcknowledgementTL is supported by a scholarship of the Anandamahidol Foundation and CFPis supported by the Scholar Award of the Michael Smith Foundation forHealth Research.Figure 2 Kaplan Meier analysis for disease-free survival and log-rank test for comparison between groups: A, HPV-HR (P =0.05);B, HPV-16/18 (P = 0.38); C, p16 (P = 0.08); and D, cyclin D1 (P < 0.0001).Lin et al. Journal of Otolaryngology - Head and Neck Surgery 2013, 42:23 Page 6 of 7http://www.journalotohns.com/content/42/1/23Author details1Division of Otolaryngology-Head and Neck Surgery, University of BritishColumbia, Vancouver, BC, Canada. 2Integrative Oncology, BC Cancer Agency& Research Centre, Vancouver, BC, Canada.Received: 3 December 2012 Accepted: 11 March 2013Published: 19 March 2013References1. 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Yu Z, Weinberger PM, Haffty BG, et al: Cyclin D1 is a valuable prognosticmarker in oropharyngeal squamous cell carcinoma. Clin Cancer Res 2005,11:1160–6.22. Capaccio P, Pruneri G, Carboni N, et al: Cyclin D1 expression is predictiveof occult metastases in head and neck cancer patients with clinicallynegative cervical lymph nodes. Head Neck 2000, 22:234–40.23. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G: Cyclin D1 is a nuclearprotein required for cell cycle progression in G1. Genes Dev 1993,7:812–21.24. Li Y, Nichols MA, Shay JW, Xiong Y: Transcriptional repression of theD-type cyclin-dependent kinase inhibitor p16 by the retinoblastomasusceptibility gene product pRb. Cancer Res 1994, 54:6078–82.25. Rischin D, Young RJ, Fisher R, et al: Prognostic significance of p16INK4Aand human papillomavirus in patients with oropharyngeal cancertreated on TROG 02.02 Phase III trial. J Clin Oncol 2010, 28:4142–8.26. Fakhry C, Westra WH, Li S, et al: Improved survival of patients with humanpapillomavirus–positive head and neck squamous cell carcinoma in aprospective clinical trial. J Natl Cancer Inst 2008, 100:261–9.27. Ang KK, Harris J, Wheeler R, et al: Human papillomavirus and survival ofpatients with oropharyngeal cancer. N Engl J Med 2010, 363:24–35.doi:10.1186/1916-0216-42-23Cite this article as: Lin et al.: Cyclin D1 overexpression is associated withpoor prognosis in oropharyngeal cancer. Journal of Otolaryngology - Headand Neck Surgery 2013 42:23.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitLin et al. 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